In our Protein Engineering studies we use site-directed and random mutagenesis based on PCR, molecular modeling and computational bioinformatics studies of structure function relationships. A strategy was developed to obtain complete genes of new enzymes, which includes screening by PCR mediated by Consensus-Degenerate Hybrid Oligonucleotide Primers (CODEHOP) and an improved genome walking method. We will continue our modeling on the “cryophilicity” of cold active enzymes through a recently established collaboration with the group of Prof. Sir Tom Blundell at the University of Cambridge, which includes crystallography and NMR studies of cryophilic enzymes.
Our group is strong in the rational design of proteins and the study and analysis of information regarding structure-function relationships in protein molecules. For the large-scale prediction of the effect of mutations, we have developed a successful methodology (the MOSST algorithm) that takes into account the contribution of each amino acid to the general structure of the protein through their main physicochemical properties. The algorithm determines which are the positions that could be mutated with or without altering the common characteristics of the protein family, and gives mutagenesis estimations related to the possibility of whether a given amino acid change would have deleterious effects on the protein. We have applied these tools to guide the selection of mutations in protein engineering strategies to improve mesophilic and cryophilic enzymes with practical applications (glucanases, trypsins). Our strong collaboration with the group of Prof. Blundell in Cambridge also focuses on the use of this methodology to discover what are the mechanisms underlying the effect of mutations in cancer-related proteins, especially the von Hippel-Lindau tumour suppressor (pVHL), the thyroid peroxidase (TPO) and the succinate dehydrogenase (SDH) complex, and how can we take this information into account to help in decision-making related to clinical practice, treatment and prognosis of clear-cell renal cancer, pheochromocytoma, von Hippel-Lindau disease and thyroid pathologies. In the biofuels area, new enzymes have been found and generated, which have been improved for more efficient hydrolysis of lignocelluloses for bioetanol production.